The effect of salinomycin on Salmonella, Campylobacter and the intestinal microflora in experimentally infected broiler chickens

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1 The effect of salinomycin on Salmonella, Campylobacter and the intestinal microflora in experimentally infected broiler chickens C. H. JOHANSEN, L. BJERRUM, M. LUND and K. PEDERSEN* Danish Institute for Food and Veterinary Research, Hangøvej 2, DK-8200 Aarhus N, Denmark. Corresponding author: The effect of the ionophore coccidiostat salinomycin on infections with Salmonella or Campylobacter was investigated in two experimental infections studies. In addition, the effect on the natural populations of Clostridium perfringens and lactobacilli in the intestine was examined. In both infection studies, chickens were divided into six groups and kept in separate isolators from day-old. Two groups of birds received salinomycin in their feed (75 mg/kg) from day 8, whereas the remaining four groups were given feed without any antibiotics or coccidiostats. At day 14 chickens were infected with approximately 10 9 cfu of either Salmonella Typhimurium or Campylobacter jejuni. Control birds were given sterile saline. Once a week 6 birds from each group were killed and intestinal bacteria were enumerated by cultivation from ileum and caecum. We found that chickens given salinomycin gained significantly more weight than control birds. Lactobacilli were found in high amounts in all samples but no effect of salinomycin on lactobacilli counts was detected. Likewise, we found no effect of salinomycin on the course of a C. jejuni infection. However, a significant reduction of the numbers of C. perfringens, was observed in the groups receiving salinomycin. Keywords: broiler chickens; intestinal microflora; salinomycin; campylobacter; salmonella Introduction Ionophore coccidiostats are widely used to control coccidiosis in poultry. In addition to their anticoccidial effects, these compounds also possess antimicrobial activity to certain bacteria, most notably to Clostridium perfringens. Previous investigations on susceptibility of C. perfringens have invariably demonstrated MIC values to ionophore coccidiostats, such as monensin, salinomycin and narasin that were much lower than prescribed in-feed dosage ranges (Devriese et al. 1993, Johansson et al. 2004), and in vivo studies have demonstrated inhibitory activity on C. perfringens (Elwinger et al. 1992). C. perfringens is the causative agent of necrotic enteritis and cholangiohepatitis in poultry. Although these compounds have never been approved specifically for prevention of necrotic enteritis, ionophore coccidiostats are now, after the banning of antimicrobial growth promoters, likely to be the most important components in the control of C. perfringens. Ionophore coccidiostats affect the intestinal microflora in a way, which is poorly understood. Although these compounds seem to have little direct effect on zoonotic pathogens, such as Campylobacter and Salmonella, it is relevant to investigate whether they may have an indirect effect on the colonisation and excretion of such organisms by affecting the microbial ecological balance in the intestinal tract. The present investigation was undertaken in order to study the impact of salinomycin on the intestinal flora of chickens, in particular C. perfringens and on the course of an infection with Salmonella Typhimurium or Campylobacter jejuni.

2 Materials and Methods Animals and experimental design The chickens used in the experimental infection studies were conventional broiler chickens (Ross 208) of mixed sex, purchased as day-old from a local hatchery (DanHatch A/S, DK). The chickens were transferred directly to the experimental unit, where they were housed in isolators (Montair Andersen B.V. HM 1500, The Netherlands). Transport-boxes together with feed and water samples from each isolator were analyzed for the presence of Campylobacter and Salmonella. Additionally, cloacal swabs from 5 randomly selected day-old chickens from each isolator were analysed by cultivation. In both experimental infection studies, the chickens were divided into 6 experimental groups, according to Table 1. The 2 x 6 groups, were kept in separate isolators where the chickens had access to feed and water ad libitum. They were fed a conventional wheat based broiler feed without antimicrobial additives (Danish Institute of Agricultural Sciences, Foulum, Denmark). The diet of group 1 and 2 was supplemented with the ionophore coccidiostat salinomycin (75 mg/kg) from day 8 and onwards (Table 1). At day 14 and 15 respectively, all chickens from group 1 to 4, were orally infected with approximately cfu of C. jejuni strain DVI-sc181 or S. Typhimurium DT 110 strain which is rifampicin resistant. The strains were originally isolated from chicken faeces and S. Typhimurium was made rifampicin resistant in our laboratory. The control groups (group 5 and 6) were given a 0.9% NaCl solution. The inoculation was carried out inside the isolators by individual crop instillation of 500 µl of a bacterial suspension or a NaCl solution respectively, using a 1 ml syringe with an attached flexible tube. Table 1. Illustrating the different treatments of the experimental groups. Experiment 1 Experiment 2 Isolator/ Salinomycin C. jejuni Salinomycin S. typhimurium Group - from day 8 - at day 14 -from day 8 - from day Sampling and culture of samples In the first experimental infection study with C. jejuni, samples were taken at day 7, 13, 16, 23, 30 and 36 and in the second study with S. Typhimurium, samples were taken at day 5, 12, 19, 26 and 35. At each sampling 6 birds were removed from each isolator and killed by decapitation. Immediately after this, the gastrointestinal tract was excised and the contents of the ileum and caecum were collected and placed on ice. The intestinal contents from 3 chickens were pooled by segment before further analysis. Quantitative cultivation of C. jejuni was carried out from caecal and ileal contents of the chickens at each sampling. The samples were diluted in 10-fold steps in buffered peptone water (BPW: Merck ) and quantified by the spread-plate method on mccda (Oxoid, Basingstoke, UK). mccda plates were incubated microaerophilic at 42 o C for 48 hours In order to quantify the S. Typhimurium infection, 10-fold dilutions of caecal and ileal contents in buffered peptone water were spread on Rambach agar with rifampicin (50 µg/ml) and incubated aerobically at 37 o C. For the control group Rambach agar without rifampicin was used. Colonies were counted after hours.

3 Finally, in both experiments, Clostridium perfringens were counted as black colonies surrounded by a precipitation zone on Tryptose-sulphite-cycloserine agar (Granum, 1997) containing egg-yolk, after anaerobic incubation at 37 o C in 24 hours. Lactobacilli were enumerated on Rogosa agar (Merck 5413), after anaerobic incubation at 37 o C in 48 hours. The experiment was made in accordance with the guidelines from the Danish Ministry of Justice with respect to animal experiments. Results and discussion A stable infection with C. jejuni was established in the ileum from day of infection throughout the experiment. This is in accordance with previous experiments (Johansen et al. 2006). There were no significant differences in ileal counts of Campylobacter between groups given salinomycin and groups without salinomycin. Likewise, there were no differences in counts of lactobacilli between the 3 treatments. Thus, salinomycin seems to have no effect on lactobacilli or a Campylobacter infection and Campylobacter had no effect on counts of C. perfringens or lactobacilli (Table 2). These observations are in accordance with results reported by Bolder et al. (1999). However, previous investigations in our laboratory have shown that a Campylobacter infection may have an influence on the composition of the microflora. Further investigations are now in progress on the present material. a) Chicken Meanweight (kg) 2,50 2,00 1,50 1,00 0,50 Salinomycin + C. jejuni C. jejuni Control 0, Age (days) b) Chicken Mean Weight (kg) 2,50 2,00 1,50 1,00 0,50 Salinomycin + S. typhimurium S. typhimurium Control 0, Age (days) Figure 1. Meanweight of the chickens in the two different studies. a) In the experimental infection using C. jejuni. b) In the experimental infection using S. typhimurium.

4 Table 2. Bacterial counts (log10 cfu/g) from the ileal content of the chickens. Group 1 4 was infected with C. jejuni as 14-day-old. Two of these groups (1 2) were given feed containing the ionophore coccidiostat, salinomycin from day 8. Group 5 6 was kept as control chickens. The detection limit for bacterial counts was Campylobacter Clostridium perfringens Lactobacilli Age (days) Group Treatment 1-1 Salinomycin + nd nd nd 3.23 nd nd nd nd < Campylobacter nd nd nd 3.28 nd nd nd nd < Salinomycin + nd nd ne nd nd nd 2.70 < Campylobacter nd nd nd nd nd nd 2.30 <5 ne Campylobacter nd nd 6.73 ne ne < nd nd < Campylobacter nd nd < nd nd nd 3.94 < Control nd nd nd nd nd nd < nd nd nd nd nd nd < Control nd nd nd nd nd nd nd nd nd < nd nd nd nd nd nd nd nd 5.04 < nd = none detected. ne = not examined.

5 Table 3. Bacterial counts (log10 cfu/g) from the ileal content of the chickens. Group 1 4 was infected with S. typhimurium as 15-day-old. Two of these groups (1 2) were given feed containing the ionophore coccidiostat salinomycin from day 8. Group 5 6 was kept as control chickens. The detection limit for bacterial counts was Salmonella Clostridium perfringens Lactobacilli Time (day) Group Treatment 1-1 Salinomycin + nd nd nd nd nd nd S. typhimurium nd nd 2.78 nd nd nd nd nd Salinomycin + nd nd nd nd nd 3.48 nd nd nd nd S. typhimurium nd nd 2.00 nd nd 3.28 nd nd nd nd S. typhimurium nd nd 2.00 nd nd nd nd nd 2.85 nd nd nd < nd S. typhimurium nd nd 3.61 nd nd nd < nd nd nd nd nd nd Control nd nd nd nd nd nd nd nd nd nd nd Control nd nd nd nd nd nd nd nd nd nd nd = none detected.

6 In the present experiment, a stable infection with Salmonella was not achieved. This is in contrast to previous investigations (Bjerrum et al. 2003). In the Salmonella experiment, there was no statistical difference in counts of C. perfringens between infected and uninfected groups. Likewise, there were no statistical differences in counts of lactobacilli between the three treatment groups (Table 3). The effect of salinomycin on C. perfringens was dramatic. Most counts of C. perfringens in salinomycin were below detection level (Table 2 and 3). In groups not receiving salinomycin, count of C. perfringens were on average in the level c.f.u./g, and ranging from below detection level to These figures are somewhat lower than recorded in previous experiments (Pedersen et al. 2003). The impact of salinomycin on growth was very pronounced (Figure 1). In the Salmonella experiment, the groups given salinomycin in the feed had a 45% higher body weight at day 35 compared to the groups not given salinomycin. In the Campylobacter experiment the same figure was 62%. This extremely high benefit from using salinomycin in the feed, which exceeds experiences from in vivo studies, is difficult to explain. An inhibitory effect of C. perfringens in untreated groups may be part of the explanation, but it seems more likely that other factors play a role, too. Investigations are in progress to elucidate this in more detail. References BJERRUM, L., ENGNERG, R.M., LESER, T.D., JENSEN, B.B., FINSTER, K. and PEDERSEN, K. (2006) Microbial community composition of the ileum and cecum of broiler chickens as revealed by molecular and culture based techniques. Poult. Sci. (in press). BJERRUM, L., ENGNERG, R.M. and PEDERSEN, K. (2003) Infection model for Salmonella typhimurium DT110 in day-old and 14-day-old broiler chickens kept in isolators. Avian Dis. 47: BJERRUM, L., PEDERSEN, K. and ENGNERG, R.M. (2005) The influence of whole wheat feeding on salmonella infection and gut flora composition in broilers. Avian Dis. 49: BOLDER, N.M.,WAGENAAR, J.,PUTIRULAN, P.P., VELDMAN, K.T. and SOMMER, M. (1999) The effect of flavophospholipol (Flavomycin) and salinomycin sodium (Sacox) on the excretion of Clostridium perfringens, Salmonella enteritidis, and Campylobacter jejuni in broilers after experimental infection. Poult. Sci. 78: DEVRIESE, L.A., DAUBE, G., HOMMEZ, J., HAESEBROUCK, J. (1993) In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics. J. Appl. Bacteriol. 75: GRANUM, P.E. (1997) Clostridium perfringens. Determination in Foods. Nordic Committee on Food Analysis. No. 95, 3 rd ed. JOHANSEN, C.H., BJERRUM, L., FINSTER, K. and PEDERSEN, K. (2006) Effects of a Campylobacter jejuni infection on the development of the intestinal microflora of broiler chickens. Poult. Sci. 85: JOHANSSON, A., GRECO, C., ENGSTRÖM, B.E. and KARLSSON, M. (2004) Antimicrobial susceptibility of Swedish, Norwegian, and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes. Vet. Microbiol. 99: PEDERSEN, K., BJERRUM, L., NAURBY, B. and MADSEN, M. (2003) Experimental infections with Clostridium perfringens strains in broiler chickens using isolator facilities. Avian Pathol. 32: